Overview: Our research program addresses basic molecular and physiological processes of nociceptive transmission in the central and peripheral nervous systems and new ways to effectively treat intractable pain. The molecular research is performed using animal and in vitro, cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that innervate the skin and deep tissues and their connections in the dorsal spinal cord, which is the first site of synaptic information processing for pain. Our research has identified the DRG and spinal cord as loci of neuronal plasticity and altered gene expression in persistent pain states. The mechanisms of transduction of physical pain stimuli are also under investigation through examination of events in damaged or inflamed peripheral tissue and using reductionistic approaches such as cloned thermal and chemo-responsive ion channels expressed in heterologous cell systems or naturally expressed in primary cultures of dorsal root ganglion. Our goals are (1) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (2) mechanisms underlying human chronic pain disorders, and (3) to use this knowledge to devise new treatments for pain.[unreadable] New Treatments for Pain: We address the new treatment goal by a translational research and human clinical trials program aimed at developing new analgesic treatments for severe pain. The current approach is based on our studies of pain transduction through the vanilloid receptor 1 (TRPV1). This molecule is a heat-sensitive calcium/sodium ion channel and converts painful heat into nerve action potentials by opening the pore of the ion channel, which then depolarizes pain-sensing nerve endings and triggers an action potential that is conducted to the spinal cord. Capsaicin, a vanilloid chemical and the active ingredient in hot pepper, also stimulates channel opening. We use a very potent vanilloid called resiniferatoxin (RTX) to prop open the ion channel, thereby causing calcium cytotoxicity and death of a specific class of pain-sensing neurons. This proved to be a very effective means of pain control in several pre-clinical models and via several routes of administration. Some routes will lead to cell death (e.g. intraganglionic injections) other routes do n. We have established an inter-institute working group with NIDA's Division of Pharmaco-Therapeutics and Medical Consequences of Drug Abuse to bring this novel treatment to human clinical trial. The working group consists of experts on chemistry and manufacture, toxicological, neurobiological, medical, and regulatory affairs as well as pain management specialists and pharmacologists from our group. We have also established procedures for obtaining the natural product from which the active drug is extracted and procedures for isolation, purification and formulation of the drug product that are compliant with Food and Drug Administration (FDA) regulations. We are presently finalizing the toxicology study and preparing the Investigational New Drug Application for submission to the FDA. We are also finalizing the human clinical protocol with the Institutional Review Board. The RTX cell deletion treatment will first be tested for its ability to control cancer pain in patients with advanced disease. If it is safe and effective, we shall conduct a second protocol for treatment of head and neck cancer and then work on controlling other chronic pain conditions such as trigeminal neuralgia, arthritis and neuropathic pain.[unreadable] Clinical Pain Mechanisms: Chronic neuropathic pain conditions, either in the body or the oro-facial region, are difficult to treat effectively. They are also difficult to explore via pre-clinical investigations since informative animal models with clearly predictive value are not available. One particularly difficult human neuropathic pain problem is Complex Regional Pain Syndrome (CRPS, formerly called reflex sympathetic dystrophy). This syndrome arises subsequent to a nerve injury or trauma and is over-represented in women. We are testing the hypothesis that an autoimmune response is provoked to one or more proteins from small diameter pain-sensing axons (C-fiber or A-delta neurons). We have established a very sensitive assay to test for the presence of autoantibodies to tissue proteins in general. We are applying the method to measure antibodies against primary afferent neuronal proteins in the serum of patients with CRPS as well as other neuropathies and nervous system disorders. For example we are examining patients with other autoimmune diseases that exhibit neuropathic pain to determine if there is an overlap in antigen profiles indicative of a common denominator or general mechanism. Other controls involve patients that have experienced a nerve injury but do not develop CRPS to establish clinical specificity and a series of positive and negative controls . One autoimmune disorder we are investigating is Sjogren's syndrome (SS). Approximately 30 % of patients develop a small fiber painful neuropathy, which frequently progresses to a mixed large and small fiber neuropathy. Thus, C-fibers appear to be an autoimmune target in this syndrome, interestingly, like CRPS, SS also is manifested predominantly in women. We are collaborating with the Gene Therapy and Therapeutics Branch, NIDCR to examine sera from Sjogren's patients to test against our antigen panel. We have had great success in initial studies with known antigens in SS and other autoimmune disorders (Stiff Person Syndrome and Type 1 Diabetes). We have adapted the assay to microtiter plate format and to run on our robotic workstation. As time progresses, we shall expand the analyses other cohorts of chronic pain patients to establish whether the underlying mechanisms are generalized or specific to these types of nerve injury-induced neuropathies. Multiple antigen profiling yields a new progressive level of understanding for complex human disease states.[unreadable] Basic Pain Mechanisms: Underlying the translational studies are our investigations of molecular regulation of gene expression, neuronal function, behavior, and mechanisms of pain transduction. We are systematically investigating the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal spinal cord. This approach provides a comprehensive and informative view of nociceptive process. The dorsal root ganglion is quite small but its analysis is made possible by the extensive use of reverse-transcription-PCR, which allows us to make multiple measurements on such small tissue samples. Our studies reveal the dynamic modulation of gene expression at all three steps in a more complex fashion than previously hypothesized. We have examined novel molecules as well as neuropeptide, cytokine and chemokine expression and identified prominent roles for new, key molecules with distinct combinatorial patterns of expression among the three tissues. The functional implications of our studies on cytokines suggest a new role for monocyte chemoattractant protein 1 in nociceptive DRG neurons. We are trying to evaluate the effects of C-fiber activation and cytokines on immune cell dynamics and endothelial responses as the triggers for neurogenic inflammation and plasma extravasation in real time. We hope to obtain a fundamental understanding of the relationships between tissue damage, inflammation and pain sensation. In a broader framework, these studies fundamentally explore the molecular basis of synaptic plasticity. We hypothesize modularity in neuronal responses to new levels of synaptic or pharmacological input that will be relevant not only to pain, but also to situations such as learning, neurological disorders like epilepsy, and drug abuse